Hepatic neddylation deficiency triggers fatal liver injury via inducing NF-κB-inducing kinase in mice

The conjugation of neural precursor cell expressed, developmentally downregulated 8 (NEDD8) to target proteins, termed neddylation, participates in many cellular processes and is aberrant in various pathological diseases. Its relevance to liver function and failure remains poorly understood. Herein, we show dysregulated expression of NAE1, a regulatory subunit of the only NEDD8 E1 enzyme, in human acute liver failure. Embryonic- and adult-onset deletion of NAE1 in hepatocytes causes hepatocyte death, inflammation, and fibrosis, culminating in fatal liver injury in mice. Hepatic neddylation deficiency triggers oxidative stress, mitochondrial dysfunction, and hepatocyte reprogramming, potentiating liver injury. Importantly, NF-κB-inducing kinase (NIK), a serine/Thr kinase, is a neddylation substrate. Neddylation of NIK promotes its ubiquitination and degradation. Inhibition of neddylation conversely causes aberrant NIK activation, accentuating hepatocyte damage and inflammation. Administration of N-acetylcysteine, a glutathione surrogate and antioxidant, mitigates liver failure caused by hepatic NAE1 deletion in adult male mice. Therefore, hepatic neddylation is important in maintaining postnatal and adult liver homeostasis, and the identified neddylation targets/pathways provide insights into therapeutically intervening acute liver failure.


NCOMMS-21-06607 Review
In this manuscript, the authors investigate a role for NAE1 and neddylation in human ALF disease. Through the use of conditional deletion of NAE1 in the embryo (Alb-CRE) and the adult knockouts, they show that reduction of neddylation leads to phenotypes similar to ALF, such as hepatocyte injury and death, liver inflammation and fibrosis. Gene Expression from both the embryonic knockout (LKO) and adult knockout show very similar responses in gene expression, as well a,s pathways that point increased oxidative stress and mitochondrial dysfunction which are thought to contribute to the liver phenotype.
They also show the decrease in mature hepatocyte gene expression and increase in genes and proliferation (Ki67+) reflecting BEC/progenitor following injury. This suggests a role for cellular reprograming and/or regeneration in the pathophysiology of this phenotype. They also demonstrated a cell autonomous effect in CRISPR NAE1 knockouts in HepG2 cells and with neddylation inhibitor MLN in vitro with similar effects on gene expression.
Mechanistically, they show a connection between Neddylation and NF-kB inducing kinase (NIK) stability. NIK is highly regulated by ubiquitination and degradation and is stabilized by a subset of TNF receptor family members upstream of alternative NF-kB signaling. They show a similar stabilization and activation of NIK in the absence or inhibition of neddylation enzymes leading presumably to p100 phosphorylation and increased p52 processing. They also demonstrate a benefit with oxidative stress inhibition (NAC) on the mouse liver phenotype.
Comments: (1) Only figure 6 refers to 3 independent experiments with representative images and plots of data. Would the authors please clarify how many times each set of observations was performed including embryonic and adult knockout cohorts? (2) The authors suggest that there is significant perturbation of neddylation enzyme expression in two ALF patient datasets, with NAE1 being significantly downregulated. If they have access to ALF patient samples, measuring NAE1 expression for themselves would be preferable and would make a nice figure 1 A to set up the manuscript for why they have targeted NAE1. Authors may not have access to these human samples, but measuring NAE1 expression in multiple patient samples may raise the quality of the data and further support their reference to the phenotype in this disease as Figure 1A rather than in the supplement. Seeing the spread of the data in the patient population compared with control would be important.
(3) In figure 1 A, the authors show a nice reduction in N8-Conj in the KO P7 and p10, but this affect goes away by P14. Would the authors please comment on why this might be. Are their compensatory mechanisms that occur over time? Do other neddylation enzymes also use N8 as a substrate? (4) In figure 1, the liver injury, inflammation and fibrosis are clear. Curious whether they looked at hydroxyproline levels and/or collagen gene expression in the embryonic knockout? Collagen genes were measured in the adult knockout. (5) In reference to figure 1F, they refer to plasma ALT as a measure of liver function. While that is correct for plasma bilirubin, plasma ALT is a measure of hepatocyte damage and death not function. (6) The quality of the plot in figure 1H is poor. It is hard to see the dynamic range of the effect when the symbols are so low against the axis. This plot should be redone so that we can effectively see the fold change in expression of these genes. (7) Would the authors speculate on why they observe steatosis in the adult but not in the embryonic knockout? (8) In figure 3D, the reduction of mature hepatocyte markers and increase in BEC/progenitor markers is interesting. Is this due to mature hepatocyte death, reprogramming of some cells to progenitors or expansion of existing progenitors in the liver? It is hard to tell whether this effect is due to cellularity differences in the bulk tissue sample. Since this is RNA from bulk tissue, have the authors thought about doing single cell RNAseq to better tease out the cell populations that are present in these mice? The Ki67+ staining does suggest increased progenitor activation following injury, but given the current analysis it is hard to tell what they are and where they are coming from especially since the increase in progenitor activation (if it is occurring) does not keep up with the hepatocyte damage in this model. Is this an example of "frustrated" progenitors that are not capable of further differentiation following activation? Single cell RNAseq may better define known progenitor populations in the liver and give a more complete data set. (9) Figure 4 shows a nice correlation between the embryonic and adult knockouts. Have the authors correlated these genes with the ALF data sets to see how relevant their model is to human AFL disease beyond just NAE1 expression and other neddylation enzymes? This would strengthen the relevance of the paper to human disease. (10) Figure 5 F Y-axis should start at 0 as not to overemphasize the effect between groups. (11) Figure 6 A, the authors should consider plotting C/ebpa and Hnf4a differently as to be able to see the fold change between groups. Although referred to as significant it is too small a difference as plotted to see effectively. (12) Regarding Figure 6 A and B, would the authors please comment relatively limited effect in reduction of mature hepatocyte markers C/ebpa and Hnf4a. The effect between the two appears different between primary hepatocytes and HepG2 cells. This is less dramatic than the gene expression in bulk tissue. The authors should comment on why that might be and if this truly represents "reprogramming" to a progenitor like state or just transient gene expression differences. Again, single cell might give a more wholistic representation of what is going on here. (13) The authors clearly show the effect of neddylation on NIK stability. What is lacking in this dataset are the downstream effects of increased NIK stability/activity. The effect is mostly done with overexpressed NIK protein. Overexpression of NIK can have effects that may be different from endogenous levels of regulated NIK. Endogenous NIK can be hard to detect and pull down, but have they tried to look at the same effect on endogenous NIK? Have the authors thought about using proteomic approaches to observe neddylated and ubiquitinated NIK peptides? (14) Figure 7A, the results appear inconsistent. Would the authors please comment on how many times these results were observed and how many individual mice were tested by western? The day 24 p52 blot is not very good. Is this whole tissue/cell lysate or nuclear extract? Nuclear extracts generally reflect a clearer increase in p52. Also curious what the total p100 bands look like above the p52 band. Do they observe a reduction in the p100 band given the rather modest effect on p52 in this case? (15) In figure 7, MLN inhibition leading to reduced neddylated NIK is not very strong. I do acknowledge that the IP western detection is not very quantitative, however the reduction in figure 7D & E is not very strong. Did the authors titrate the amount of MLN used and test earlier timing of the MLN treatment post HepG2 transfection to get a more optimal result? Does the residual neddylation represent the amount of NIK neddylated prior to MLN treatment? Would the authors speculate on the rate of turnover of neddylated NIK. (16) The authors make nice case for the effect of reduced oxidative stress (NAC treatment) on the NAE1 knockout phenotypes. Reduction in liver injury, inflammation, and fibrosis, gene expression as well as the mature hepatocyte vs BEC/progenitor genes, with clear controls to show reduction in markers of oxidative stress (supplement). What is lacking is the same rigor of working out the role of NIK in these phenotypes. Given the importance the authors are putting on the NIK neddylation connection, the same depth of investigation into the effect of NIK knockout or inhibition on the NAE1 / MLN phenotype in vivo would be important. The authors do point to the what has been shown in the literature about NIK transgene on liver injury / disease phenotypes, however, what is lacking is the direct effect of NIK inhibition / knockout on neddylation deficient phenotypes, such as hepatocyte injury and death, mature vs progenitor gene expression balance, oxidative stress, etc. (17) Authors do show a 50% reduction in HepG2 apoptosis (Casp3) with NIK inhibitor B022 induced by TNFa similarly to NAC, but do not go beyond that. If the B022 compound is the one that I am familiar with from Amgen, the compound lacks significant potency as well as has selectivity limitations. Have the authors tried treating with B022 to see if NIK is required for the effect of NAE1 knockout or MLN on HepG2 induction of progenitor genes and repression of mature hepatocyte genes?
Overall, this manuscript demonstrates a clear effect of neddylation deficiency on liver injury and disease. The following should be addressed for acceptance: (1) The authors show a reduction in mature hepatocyte markers and an increase in progenitor markers and suggest that there might be cellular reprogramming going on in response to liver injury. Rather than relying on bulk mRNA changes, the authors should use single cell RNAseq to identify cell populations that are present at the time of disease which would be greatly informative here.
(2) A correlation between knockout RNAseq data sets with ALF correlated genes would greatly strengthen their story and the proposed connection between NAE1/Neddylation and ALF liver disease.
(3) More correlation or efficacy data with NIK knockout or inhibition in the context of NAE1 knockout induced liver disease. While the NIK biochemical results on neddylation are intriguing, the proposed hypothesis of the relationship to human disease requires more direct links between NIK activity and downstream alternative NF-kB signaling with neddylation induced disease and possible relationship to human ALF would be important to see if accepted for Nature Communications.

RESPONSES TO REFEREES
We thank the Editor and Reviewers for the constructive comments. We have significantly improved the manuscript according to the helpful suggestions. We combined the original Fig. 4 and Fig. 5 to a new Fig. 4. We performed proteomics analysis and identified the neddylation sites of NIK. These sites were mutated to test their involvement in mediating NIK ubiquitination and protein stability. We further examined the contribution of NIK signaling to neddylation deficiency-induced acute liver failure (ALF) both in vitro and in vivo using the NIK specific inhibitor B022. In addition, we also investigated whether blocking TNF or apoptosis/necroptosis signaling pathways could ameliorate NAE1 deletion-induced ALF, respectively. These data are included into new Fig. 6, Fig. 7 and Supplemental Figs. S7 and S8. Below is the point-by-point response to the comments.
Reviewer #1 (Remarks to the Author): The authors show that perinatal or adult impairment of Neddylation causes liver failure. The derangements are very broad and challenge the interpretation of which are cause or effect. The Neddylation effect on NIK expression is novel but the contribution to ALF is unclear.
1 -Although the KEGG analysis modestly point to TNF signaling (4B), the expression of TNF was even less striking (1H and 2F). The only strong support for importance is the HepG2 experiment which shows sensitization to TNF but many hepatic insults are known to sensitize to TNF. The effect of TNF blocker (e.g. infliximab) or receptor knockdown in vivo should be examined.

Response:
We agree with the reviewer. In both P14 LKO and D21 Cre livers, the upregulated genes involved in TNF signaling pathway include inflammatory cytokines (Ccl2 and Lif), chemokines (Cxcl10), interferon gamma inducible protein 47 (Ifi47), transcription factor (Jun), necroptosis marker (Mlkl) and adhesion molecule (Sele). These genes are all downstream of TNF signaling. We did not find that Tnf was significantly upregulated in neonatal NAE1-deleted liver at P14, a relatively early stage of disease progression (Fig. 1h), but it was elevated in D24 AAV-Cre livers (Fig. 2f). To directly address whether TNF signaling contributes to neddylation deficiency-induced liver injury, we treated D10 AAV-Cre mice with Infliximab (the TNF blocker) every other day and monitored their survival compared to vehicletreated AAV-Cre mice. Infliximab only produced a trend toward improving the survival in NAE1 deletioninduced acute liver failure (Please see Supplemental Fig. S8h). These data suggest the potential involvement of TNF signaling in neddylation deficiency-induced ALF. However, divergent inflammatory pathways are linked to neddylation deficiency. Blocking TNF alone is not sufficient to reduce mortality.
2 -Evidence of apoptosis and necroptosis is presented. Each could be triggered by TNF but not likely in the same cells at the same time. Need IHC to assess which cells are involved and if there are zonal differences in apoptosis or necrosis.

Response:
We agree with the reviewer. Our human liver pathologists carefully re-examined liver histology. Both P14 LKO and D24 AAV-Cre livers demonstrated no zonal differences in apoptosis.
While the necrosis was only occasionally present in P14 LKO livers, a centrilobular (acinar zone 3) necrosis in livers of D24 AAV-Cre mice was observed. Please see the revised histological descriptions in Page 7, Results section. We performed additional IHC staining for RIPK3, a critical mediator of necroptosis, and confirmed increased RIPK3 expression concentrated in zone 3 of the livers of D24 AAV-Cre mice. We have added these data to Supplemental Fig. S3a in the revised manuscript.
3 -The impact of pancaspase inhibitor (e.g. Z-VAD) and RIPK1 inhibitor (Nec-1s) or both should be assessed to determine which cell death mechanism accounts for the liver failure. though oxidative stress appears to account for some.

Response:
We completely agree with the reviewer. Neddylation participates in diverse cellular processes and pathophysiological events. Our findings present the first line of evidence that neddylation is essential to normal liver function by regulating hepatocyte oxidative stress, mitochondrial dysfunction, NIK signaling etc. We think that dysregulation of these critical cellular processes collectively lead to a rapid development of fatal liver failure. Given the power of neddylation in liver, simply blocking one single pathway (NIK, TNF or cell death) may not be sufficient to rescue the fatal phenotype seen in NAE1-deficient mice. Our findings further demonstrate that antioxidants are the most effective treatment for ALF in AAV-Cre mice, similar to its efficacy in human ALF. This information have been included in Paragraph 1, Discussion section, 5 -The mechanism of oxidative stress certainly may be linked to GSH depletion and mitochondrial impairment but cause and effect are not clearly defined. The mechanistic link between NIK and the biochemical pathologic events needs to be explored more deeply. As presented this is not directly elucidated.

Response:
In response to this comment, we performed proteomic analysis in HEK293T cells following co-expression of NIK-HA with FLAG-NEDD8, and identified four potential neddylation sites of NIK. These sites were mutated and shown to be important in mediating NIK ubiquitination and protein stability (new Fig. 6f-I, and Supplemental Fig. S7c-e). We further demonstrated that NIK signaling contributes to neddylation deficiency-induced ALF both in vitro and in vivo using the NIK specific inhibitor B022. B022 alleviates MLN induced fetal reprogramming and desensitizes hepatocytes to TNF triggered apoptosis. We also administrated B022 daily to D10 AAV-Cre mice. A 50% inhibition of NIK signaling was achieved but this did not improve overall mortality in AAV-Cre mice. However, B022 treatment produced a trend towards reducing the expressions of progenitor and inflammatory genes and the necroptosis and apoptosis-related proteins in AAV-Cre livers. This suggests that NIK is In this manuscript, the authors aim to clarify the role of neddylation deficiency in the regulation of acute liver injury. They reported that hepatic-specific inhibition of neddylation by NAE1 deletion caused hepatocyte damage, mitochondrial dysfunction, and excessive fetal liver reprogramming. Besides, the authors identified NIK as a novel NEDD8 target. The authors also reported that antioxidant therapy partially rescued NAE1 deficiency-induced ALF. Although the topic of the research and some of discoveries the authors presented are interesting, many of data are not solid enough to support the conclusions. Some of major issues that need to be addressed are listed below 2. It is well known that neddylation deficiency causes mitochondrial dysfunction, apoptosis and cell death. The authors also showed that hepatocyte-specific neddylation deficiency caused hepatocyte damage, mitochondrial dysfunction, and excessive fetal liver reprogramming. Are the mechanisms of these phenotypes the same as the reported mechanisms of Neddylation inhibition, such as activating intrinsic and extrinsic apoptosis?
Response: MLN4924 has been reported to induce intrinsic (mitochondrial) apoptosis and activates the extrinsic (death receptor-mediated) apoptosis in cancer cells 3 . In our study, neddylation deficiency causes hepatocyte damage through induction of both intrinsic apoptosis due to mitochondrial dysfunction and extrinsic TNF-mediated apoptosis secondary to ROS or NIK mediated inflammation ( Fig. 4). While the mechanisms underlying the neddylation deficiency-induced hepatocyte reprograming remains to be further identified, a direct effect of neddylation in regulating NIK stability and function was elucidated ( Fig. 6 and Fig. 7). We also discussed the potential mechanisms of mitochondrial dysfunction and GSH depletion in neddylation-deficient hepatocytes (Please see Discussion page 19 and 20). of Results section). We found that NAE1 deletion produced a consistent activation of IκB phosphorylation. However, the net change of canonical NF-κB signaling was biphasic in vivo, with an early activation followed by a suppression (as evidenced by IκBα degradation and p65 accumulation) during the ALF progression in AAV-Cre mice. In vitro, the degradation of IκBa was not reliably inhibited in NAE1-knockdown primary hepatocytes or NAE1-deficient HepG2 cells. Therefore, neddylation deficiency leads to a consistent activation of non-canonical, but not of canonical NF-κB signaling in hepatocytes, further emphasizing a direct role of neddylation-mediated NIK signaling in ALF. figure 6A, the authors claimed that they achieved ≈50% NAE1 knockdown and suppression of neddylation in pAd-Cre. However, the N8-Conjugates had no obvious decrease upon NAE1 downregulation.

Response:
We thank the reviewer for this question. Among the N8-Conjugates, we did not observe a reduction in the Cullin sizes (N8-CULs, which was confirmed by Western blot against CUL2) in NAE1 knockdown primary hepatocytes. This could be due to a 50% NAE1 knockdown being insufficient to block neddylation of CULs. By contrast, we did observe that the neddylation conjugation of non-Cullin substrates was downregulated by NAE1 knockdown. Please see new Fig. 5a. figure 6B, in the NAE1-KO HepG2 cells, the N8-Conjugates strongly increased. The increase of N8-conjugates is quite confusing. The authors should detect the classic substrates of Neddylation (e.g. CUL1, CUL2) to further confirm the neddylation inhibition after knockdown of NAE1.

Response:
We have performed Western blots against the classic neddylation substrates, such as CUL1, 2, and 3. We found that NAE1 knockout by two different gRNAs exerted differential effects on the neddylation of CUL1-3. While the neddylation of CUL1 was inhibited only in NAE1-KO2 HepG2 cells, reduced neddylation of CUL2 and CUL3 was obvious in NAE1-KO1 HepG2 cells (Fig. R1). Due to the limited space, we only included data for CUL2 (new 6. In figure 6B, NAE1 knockdown is more efficient in KO2 compared with KO1. However, in figure S7C, the expression of NIK and p-p100 in KO1 is stronger than that in KO2.
Meanwhile, the accumulation of p52 is not obvious enough.

Response:
We appreciate the reviewer's insights. Currently we are not sure why activation of the NIK signaling pathway in two lines of NAE1 KO HepG2 cells are not dose dependent, which was the case for other signaling pathways we examined, such as YAP and hepatocyte reprograming ( Fig. S6). figure 7, the authors showed that NAE1 knockdown induced NIK accumulation, and MLN4924 treatment delayed the degradation of NIK ( Fig. 7A-B). Co-treatment MLN and BZM synergistically elevated endogenous NIK protein accumulation (Fig. 7C). Therefore, the authors claimed that neddylation deficiency stabilizes NIK protein in a proteasome-dependent manner. All these results strongly indicate NIK may be a substrate of CRL E3 ligase, but not a direct substrate of neddylation. The authors should determine whether NIK is a substrate of CRL E3 ligase.

Response:
We thank the reviewer for the valid points. NIK has been shown to be ubiquitinated by several non-CRL E3 ubiquitin ligases, such as cIAPs 4 , ZFP91 (Zinc finger protein 91) 5 , Peli1 6 and CHIP (carboxyl terminus of HSC70-interacting protein) 7 . To test whether CRL E3 ligases are involved, we obtained gRNAs against CUL1, 2, 3, 4a and 4b, respectively in an attempt to delete each CUL in HepG2 cells using a CRISPR/Cas9 strategy. We successfully obtained CUL2-KO and CUL3-KO HepG2 cells but there was no upregulation of NIK expression (these data have been included in new To pinpoint whether NIK is a direct substrate of neddylation, we performed proteomic analysis and identified the potential neddylation sites of NIK. The involvement of these sites in NIK neddylation and protein stability was also examined (Please see response to Reviewer 1 comment 5). These data clearly strengthen our conclusion that NIK is a direct substrate of neddylation. Please see the new Fig.   6 for more details.
8. In figure 7, the authors tried to prove that NIK is a neddylation substrate, however, the data provided are not sufficient to support this conclusion. Much more work is needed to strengthen the conclusion.
Besides, in figure 7D, the Flag-NEDD8 band in input in very confusing. In figure 7E, the endogenous co-IP assay result is not satisfactory. I would say the interaction is so weak that it cannot be concluded that UBC12 interacted with NIK.

Response:
We have added additional data to further prove that NIK is directly neddylated (please see response to Comment 7). In Fig. 7D (now Fig. 6c), we only presented the Western blot at the size of free NEDD8 form. While the FLAG-N8 WT could be largely conjugated to the substrates, the FLAG-N8 ΔGG was unable to conjugate, leading to the massive accumulation of FLAG-N8 ΔGG .
We have now scaled up the Co-IP assays in BZM-treated HepG2 lysates and identified a clear signal at the NIK size that co-immunoprecipitates with UBC12. These data provided direct evidence that endogenous NIK is a substrate of neddylation. Please see Fig. 6d. However, in figure S7E, the mRNA level of Mapk3k14 also seem to be upregulated, so this conclusion is not very clear. The authors did not provide the corresponding significance analysis in the bar plot or legend.
Response: Thank you for pointing that out. We have now added the significance analysis in the bar plot to show that the changes did not reach significance based on One-way ANOVA followed by 10. In Figure S6, the figure legend is not corresponding to the figure.
Response: We sincerely apologize for the mistake and have corrected the figure legend in Fig. S6. 11. In figure 3C, the C/EBPα is labeled P30. In figure 6A/6B/6D, the C/EBPα is labeled p42. What is the difference?
Response: Thank you for mentioning that. There are two isoforms of C/EBPa (p42 and p30) which play slightly different roles in liver development and function. We found that the antibody against C/EBPa (sc-61, scbt) dominantly detected the p30 form of murine source, whereas p42 was mainly detected in human samples such as HepG2 cells. Please see the source Western blot data with two different isoforms of C/EBPa labeled separately.

NCOMMS-21-06607 Review
In this manuscript, the authors investigate a role for NAE1 and neddylation in human ALF disease.
Through the use of conditional deletion of NAE1 in the embryo (Alb-CRE) and the adult knockouts, they show that reduction of neddylation leads to phenotypes similar to ALF, such as hepatocyte injury and Mechanistically, they show a connection between Neddylation and NF-kB inducing kinase (NIK) stability. NIK is highly regulated by ubiquitination and degradation and is stabilized by a subset of TNF receptor family members upstream of alternative NF-kB signaling. They show a similar stabilization and activation of NIK in the absence or inhibition of neddylation enzymes leading presumably to p100 phosphorylation and increased p52 processing. They also demonstrate a benefit with oxidative stress inhibition (NAC) on the mouse liver phenotype. Comments: (1) Only figure 6 refers to 3 independent experiments with representative images and plots of data.
Would the authors please clarify how many times each set of observations was performed including embryonic and adult knockout cohorts?

Response:
We have now included a statement on how many times of each in vivo and in vitro experiment was performed in the Statistics Paragraph of the Methodology section. We have also included specific animal numbers for each cohort experiment in each Figure legend.
(2) The authors suggest that there is significant perturbation of neddylation enzyme expression in two ALF patient datasets, with NAE1 being significantly downregulated. If they have access to ALF patient samples, measuring NAE1 expression for themselves would be preferable and would make a nice figure 1 A to set up the manuscript for why they have targeted NAE1. Authors may not have access to these human samples, but measuring NAE1 expression in multiple patient samples may raise the quality of the data and further support their reference to the phenotype in this disease as Figure 1A rather than in the supplement. Seeing the spread of the data in the patient population compared with control would be important.

Response:
We agree with the reviewer that it would be preferable to measure NAE1 expression and neddylation levels in ALF patient samples. Unfortunately, we found no such commercial or academic source for these samples. Following the reviewer's suggestion, we have plotted the overall RPKMs of NAE1 detected in the ALF patient populations from the two GSE datasets, which clearly demonstrated a reduction of NAE1 expression in ALF groups. These data have been now included as Supplemental Response: We appreciate the reviewer's insight. We think that the lack of clear differences in N8-Conj in P14 LKO liver could be due to significant infiltration by non-hepatocytes. We have included this statement into the Result section, Paragraph 2.
(4) In figure 1, the liver injury, inflammation and fibrosis are clear. Curious whether they looked at hydroxyproline levels and/or collagen gene expression in the embryonic knockout? Collagen genes were measured in the adult knockout.

Response:
We measured the expression of Col1a1 and Col1a3 genes by qRT-PCR and the results show a tendency toward higher expression in P14 LKO livers (Please see Fig. 1i). We also measured liver hydroxyproline levels. These were increased in livers of male P14 LKO mice (included as Fig. 1j), in line with the increased expression of fibrotic genes and elevated Sirius red staining. The hydroxyproline levels in adult D24 AAV-Cre livers were also higher compared to AAV-GFP mice. These new data are included in Fig. 2g. (5) In reference to figure 1F, they refer to plasma ALT as a measure of liver function. While that is correct for plasma bilirubin, plasma ALT is a measure of hepatocyte damage and death not function.
Response: Thank you very much for pointing that out. We have reworded the text accordingly.
(6) The quality of the plot in figure 1H is poor. It is hard to see the dynamic range of the effect when the symbols are so low against the axis. This plot should be redone so that we can effectively see the fold change in expression of these genes.

Response:
We have replotted Fig. 1h to make it more readable. (10) Figure 5 F Y-axis should start at 0 as not to overemphasize the effect between groups.
(11) Figure 6 A, the authors should consider plotting C/ebpa and Hnf4a differently as to be able to see the fold change between groups. Although referred to as significant it is too small a difference as plotted to see effectively.

Response:
We have replotted Fig. 6A (now Fig. 5a) accordingly. Thank you very much! (12) Regarding Figure 6 A and B, would the authors please comment relatively limited effect in reduction of mature hepatocyte markers C/ebpa and Hnf4a. The effect between the two appears different between primary hepatocytes and HepG2 cells. This is less dramatic than the gene expression in bulk tissue. The authors should comment on why that might be and if this truly represents "reprogramming" to a progenitor like state or just transient gene expression differences. Again, single cell might give a more wholistic representation of what is going on here.
Response: In response to your comment, we have now included the following sentences in Paragraph 5, Discussion section at page 21.
"Whether the neddylation-deficient hepatocytes are fully reprogrammed to the progenitors or simply present as hybrid hepatocytes for liver regeneration in vivo remains unknown. Neddylation is hyperactivated in liver cancer 9 . Therefore, it is not surprising that neddylation deficiency causes more potent downregulation of hepatocyte marker proteins in protumoral as compared to primary hepatocytes ( Fig. 5a-b). In contrast to a mild in vitro effect, neddylation deficiency leads to more severe reduction of mature hepatocyte marker genes in AAV-Cre livers in vivo (Fig. 3). Such a discrepancy could be attributed to the exacerbated hepatocyte loss triggered by initial hepatocyte injury-induced inflammation." Response: We thank the reviewer for the comments. We want to remind reviewer that the Fig. 6a and   6d, Fig. 7 and (14) Figure 7A, the results appear inconsistent. Would the authors please comment on how many times these results were observed and how many individual mice were tested by western? The day 24 p52 blot is not very good. Is this whole tissue/cell lysate or nuclear extract? Nuclear extracts generally reflect a clearer increase in p52. Also curious what the total p100 bands look like above the p52 band.
Do they observe a reduction in the p100 band given the rather modest effect on p52 in this case?
Response: In response to your comments, we have now examined NIK and its downstream signaling We have now included both p100 and p52 in all figures involving NIK signaling. Interestingly, p100, a known ubiquitination substrate 13 , is also increased in neddylation-inhibited hepatocytes and livers. This suggests that neddylation deficiency also perturbs the protein expression of p100. However, p100 overexpression itself does not directly lead to its conversion to p52, which is more reliant on the activity of NIK and the downstream phosphorylation events 14 . Therefore, our findings unequivocally point to an elevated NIK-mediated, non-canonical NF-κB signaling when neddylation is deficient.  In addition to a 50% reduction in TNF-induced HepG2 apoptosis by B022, our data shows that B022 can also partially suppress MLN-induced KRT19 gene upregulation without restoring the reduced expression of hepatocyte marker genes (C/EBPA and HNF4A) (new Fig. 7c). B022 had no effect on MLN-induced upregulation of oxidative stress genes (GCLC) or downregulation of metabolic genes (PCK1) (new Fig. 7c), which was in contrast to NAC treatment in MLN-treated HepG2 cells (new Fig.   8a). Notably, B022 was also able to suppress Krt19 upregulation in MLN-treated primary hepatocytes (new Supplemental Fig. S8f). These data are in line with the partial amelioration of progenitor gene activation in AAV-Cre livers by B022 (new Fig. 7g).
Overall, this manuscript demonstrates a clear effect of neddylation deficiency on liver injury and disease. The following should be addressed for acceptance: (1) The authors show a reduction in mature hepatocyte markers and an increase in progenitor markers and suggest that there might be cellular reprogramming going on in response to liver injury. Rather than relying on bulk mRNA changes, the authors should use single cell RNAseq to identify cell populations that are present at the time of disease which would be greatly informative here.
Response: Please see response to Comment 8.
(2) A correlation between knockout RNAseq data sets with ALF correlated genes would greatly strengthen their story and the proposed connection between NAE1/Neddylation and ALF liver disease.
Response: Please see response to Comment 9.

Reviewers' comments:
Reviewer #2 (Remarks to the Author): In the revised manuscript, the authors have addressed some of the concerns I mentioned last time. However, I would say that the some data present are still not convincing enough to draw a conclusion.
1. The authors claimed that neddylation deficiency upregulated NIK protein level, which was clearly demonstrated by the authors. The authors made efforts to confirm NIK to be a novel substrate of Neddylation and present new data in figure 6. However, the results provided in the revised manuscript are still not convincing enough to draw a conclusion that NIK is a direct substrate of neddylation.
In the revised manuscript, the authors performed proteomic analysis to identify the potential Neddylation sites of NIK. According to the methods and data provided by the authors (Fig. S7C-D), I think the identified peptides presented in Fig. S7D should be GlyGly modified peptides to characterize the potential Nedd8 modified peptides. However, these four modified peptides cannot be excluded as ubiquitinated peptides modified by endogenous ubiquitin (After trypsin digestion, both Neddylation and Ubiquitination would remain two glycine residues in the modified lysine of targets).
So far, the non-cullin substrates of Neddylation are very limited. The identification of novel non-cullin substrates of Neddylation is of great significance. In the recently published articles of non-cullin Neddylation substrates I found (PMID: 33299139 for PTEN Neddylation; PMID: 24821572 for Smurf1 Neddylation), several kinds of evidences have been provided to support the neddylation of novel targets. Therefore, the authors should provide more powerful evidence to support their conclusion.
2. In the response to comment 7 of the rebuttal letter, the authors used sgRNA strategies to knock out the CULs and successfully obtained CUL2-KO and CUL3-KO HepG2 cells. In Fig. S7A, the KO efficacy of CUL3 is not satisfactory. To determine if any of CULs is involved in regulating the stability of NIK, the authors are suggested to introduce siRNAs to knock down CULs or Rbx1/Rbx2 in HepG2 cells. That's a quick test compared to sgRNA strategy.
3. Some results the authors presented are not convincing enough, such as Fig. 5b. The authors explained that the increase signals of neddylation cullins in NAE-KO cells are caused by nonspecific effects of CRIPSR/Cas9 system. I would say the explanation given by the author is unacceptable.
Reviewer #3 (Remarks to the Author): I thank the authors for the thoughtful and thorough response to the reviewer's questions. The authors improved the western blots addressing NIK and NFkB signaling and improved concerns of plot display and comments in the results and discussion sections. They also improved the comparison of knockout DEGs with ALF data sets. The authors performed proteomic analysis linking NIK with specific sites of Neddylation and functionally linking Neddylation with NIK stability. This work represents an important contribution to the understanding of NIK regulation which is needed in the field.
Aside from a few comments to address, the authors have satisfactorily addressed my concerns for this manuscript to be published.
1. Figure S1b: I don't think plotting both data sets together is valid in this case. The two data sets should be plotted independently with statistics. This will give observation greater weight. 2. Figure 4: It is very nice to see the positive correlation between Knockout and ALF data sets. Although authors show the equation of the correlation line, pearson r and p-value would give greater statistical strength to the observation. Curious if the authors looked specifically at the strongest differentially expressed genes in the knockout data sets is the correlation stronger? Not needed for publication but could be interesting if the current overall positive correlation does not hit statistical significance. 3. Figure 7a / S8a: Authors suggest that there is an effect on p100 with Neddylation inhibition. The authors should add a comment that the possible increase in p100 is simply due to feedback loop with increased NIK stability and not necessarily directly linked to Neddylation.
Reviewer #4 (Remarks to the Author): This study by Xu C et al investigates the role of neddylation in development of functional dysfunction in the liver and though the authors have extensively revised the manuscript based on earlier reviews, a few additional issues exist.

Major points
1) The resurgence of N8-Conjugated proteins (N8-Conj) in LKO mice by P14 is rather concerning and brings into consideration compensatory mechanisms. It would be useful to have immunostained liver sections to determine source of N8-Cul in P14 LKO, rather than speculating that this comes from infiltrating cells. This is all the more relevant since different hepatic cell types seem to be responding differently to modulation of neddylation since neddylation inhibition, by using the pharmacological inhibitor, MLN4924 was shown to reduce liver injury, apoptosis, inflammation, and fibrosis by targeting different hepatic cell types (PMID: 28035772).
2) Along these lines of compensatory mechanisms, it would be useful to evaluate the effects of changes in neddylation on ubiquitylation in some more detail. Though the authors allude to it in passing in the discussion, this is especially relevant since the pathways are inter-related for regulation of protein degradation and it is possible that compensatory mechanisms could be at play, especially since it is known that ubiquitin pathways regulate liver disease as well (PMID: 34529948) and interaction between the pathways has been recognized (PMID: 22608973).
3) The authors suggest that deficiency in neddylation compromises glutathione stores to induce oxidative stress and cell death, since supplementation with NAC protects against these changes to a certain extent. However, no mechanistic data is provided on why compromised neddylation should cause a decrease in cellular GSH. Some data to confirm mechanisms of this aspect would be useful.
4) The authors show higher steatosis in adult NAE1 deleted livers. However, it was recently demonstrated that inhibition of neddylation ameliorated steatosis in NAFLD (PMID: 34153521), which is opposite of what the authors indicate. This needs to be discussed and possible explanations provided.
5) The authors need to clarify what they interpret from the RIP3K data. Active caspase 8 would cleave RIP3K and induce apoptosis, while RIP3K activation in the absence of caspase 8 functions as a molecular switch for necrosis (PMID: 23913919). However, the authors show increase in both caspase 8 and RIP3K phosphorylation by D24 in Cre mice. However, there seems to be some individual variation since the animal with lower caspase 8 has higher RIP3K and vice versa. These issues need to be clarified. 6) What is the basis for the authors statement in the discussion that antioxidants prevent acute liver failure? While NAC is an antidote in drug induced liver toxicity such as due to acetaminophen, it functions by replenishing glutathione in that context, rather than scavenging free radicals. Also, NAC prevents liver injury if given early and is unlikely to prevent liver failure if injury is ongoing. The distinction between liver injury and liver failure needs to be made clear throughout the manuscript. 7) Page 3, Line 48-The authors state "The mechanisms underlying acute liver injury are not fully understood, especially at the posttranslational level." It is assumed the authors mean acute liver "failure" since mechanisms of injury are well characterized in several systems. 8) Page 5, Line 92: The authors state "This suggests the neddylation pathway may be involved in the pathophysiology of HBV-associated ALF." This sentence is not accurate, since the mere alteration of gene expression in microarray data sets is only that-an alteration. It does not confer a cause-effect confirmation and hence it the authors should modify the sentence. 9) Were the Nae1fl/fl controls littermates? This needs to be clarified, and if so, specifically stated.

Reviewer #2 (Remarks to the Author):
In the revised manuscript, the authors have addressed some of the concerns I mentioned last time.
However, I would say that the some data present are still not convincing enough to draw a conclusion.
1. The authors claimed that neddylation deficiency upregulated NIK protein level, which was clearly demonstrated by the authors. The authors made efforts to confirm NIK to be a novel substrate of Neddylation and present new data in figure 6. However, the results provided in the revised manuscript are still not convincing enough to draw a conclusion that NIK is a direct substrate of neddylation.
In the revised manuscript, the authors performed proteomic analysis to identify the potential Neddylation sites of NIK. According to the methods and data provided by the authors (Fig. S7C-D), I think the identified peptides presented in Fig. S7D should be GlyGly modified peptides to characterize the potential Nedd8 modified peptides. However, these four modified peptides cannot be excluded as ubiquitinated peptides Therefore, the authors should provide more powerful evidence to support their conclusion.

Response:
We agree with this reviewer that identifying novel NEDD8 substrates is of great interest to the field. To date, addressing this critical question remains highly challenging, in part due to the relatively low abundance of endogenous neddylated substrates, the nature of the transient, dynamic modification, and inefficient enrichment of neddylated proteins, and as the reviewer pointed out, the difficulty in distinguishing neddylated proteins from ubiquitinated proteins. Nevertheless, we have managed to perform a series of experiments with a shortage of financial support and personnel. We have now collected substantial, compelling new evidence, which, along with those presented previously, collectively demonstrate NIK as a bona fide NEDD8 target. Below is the summary of evidence supporting NIK as a direct neddylation substrate (newly generated data were denoted as "new").

4) Although current commercially available antibodies were not able to pull down endogenous NIK
to validate its neddylation status in vivo, endogenous NIK binds to endogenous NEDD8 E2 enzyme UBC12 (Fig. 7e).
As this reviewer pointed out, we cannot rule out the possibility that the identified k-Ɛ-GG peptides are modified by Ub due to the similarity of Ub and NEDD8 modification sites. Therefore, we have toned down the conclusion by stating these four lysines as "putative" neddylation sites.
In addition, we provided new evidence supporting the neddylation of NIK promotes its ubiquitination and degradation. We found impairment of neddylation by knockdown of UBC12 or NEDD8 and MLN significantly attenuated NIK ubiquitination (New Fig 7i and Supplemental Fig. S9f).  neddylation remains to be identified. These negative data were not included in the main manuscript.
2. In the response to comment 7 of the rebuttal letter, the authors used sgRNA strategies to knock out the CULs and successfully obtained CUL2-KO and CUL3-KO HepG2 cells. In Fig. S7A, the KO efficacy of CUL3 is not satisfactory.
To determine if any of the CULs is involved in regulating the stability of NIK, the authors are suggested to introduce siRNAs to knock down CULs or Rbx1/Rbx2 hepatocyte-specific deletion of CUL3 (Figure 2 to Reviewers).    Response: We agree that our original interpretation did not make sense. In Fig. 5b, the deletion of NAE1 was validated by a significant reduction of NAE1 proteins and neddylated CUL2. Currently, we do not know why NAE1 deletion in HepG2 by CRISPR/Cas9 leads to an upregulation of unknown NEDD8positive species at the size of N8-CULs. Interestingly, we also observed elevated NEDD8-positive signals at ~45 KD in NAE1-KO Hela cells by the same CRISPR/Cas9 strategy (Figure 3 to Reviewers). To avoid confusion, we have removed the NEDD8 blot from the original Fig. 5b and the related legends.

Reviewer #3 (Remarks to the Author):
I thank the authors for the thoughtful and thorough response to the reviewer's questions. The authors improved the western blots addressing NIK and NFkB signaling and improved concerns of plot display and comments in the results and discussion sections. They also improved the comparison of knockout DEGs with ALF data sets. The authors performed proteomic analysis linking NIK with specific sites of Neddylation and functionally linking Neddylation with NIK stability. This work represents an important contribution to the understanding of NIK regulation which is needed in the field.
Aside from a few comments to address, the authors have satisfactorily addressed my concerns for this manuscript to be published.

Response:
We thank the reviewer for these very enthusiastic and positive comments on the quality and significance of our study.
1. Figure S1b: I don't think plotting both data sets together is valid in this case. The two data sets should be plotted independently with statistics. This will give observation greater weight.

Response:
We appreciate the reviewer's suggestion. We have replotted the data and demonstrated that NAE1 expression was significantly lower in ALF patients as compared to healthy subjects in each dataset. Please see the new supplemental Fig. S1b. 2. Figure 4: It is very nice to see the positive correlation between Knockout and ALF data sets. Although authors show the equation of the correlation line, pearson r and p-value would give greater statistical strength to the observation. Curious if the authors looked specifically at the strongest differentially expressed genes in the knockout data sets is the correlation stronger? Not needed for publication but could be interesting if the current overall positive correlation does not hit statistical significance.

Response:
We have now included the pearson r and p-values in Fig. 4c, which indicates statistical significance when compared to both datasets.
3. Figure 7a / S8a: Authors suggest that there is an effect on p100 with Neddylation inhibition. The authors should add a comment that the possible increase in p100 is simply due to feedback loop with increased NIK stability and not necessarily directly linked to Neddylation.

Response:
We appreciate the reviewer's suggestion. However, we could not find evidence to support that increased NIK stability impedes partial p100 proteolysis to p52 via a feedback loop. NIK activates IKKα, and then the activated IKKα phosphorylates p100, leading to its polyubiquitination by the SCF-Fbw7-E3 ligase (CRL1) complex, and proteasome-mediated degradation to p52 6 . Therefore, we modified our sentence to "The increase in p100 could be due to impaired CRL1-mediated p100 ubiquitination and degradation secondary to neddylation deficiency." We have included this comment in Results on Page 13 while describing the original Fig. 7a, now Fig. 6a.

Reviewer #4 (Remarks to the Author):
This study by Xu C et al investigates the role of neddylation in development of functional dysfunction in the liver and though the authors have extensively revised the manuscript based on earlier reviews, a few additional issues exist.
Major points 1. The resurgence of N8-Conjugated proteins (N8-Conj) in LKO mice by P14 is rather concerning and brings into consideration compensatory mechanisms. It would be useful to have immunostained liver sections to determine source of N8-Cul in P14 LKO, rather than speculating that this comes from infiltrating cells. This is all the more relevant since different hepatic cell types seem to be responding differently to modulation of neddylation since neddylation inhibition, by using the pharmacological inhibitor, MLN4924 was shown to reduce liver injury, apoptosis, inflammation, and fibrosis by targeting different hepatic cell types (PMID: 28035772).

Response:
We have performed extensive double immunofluorescence staining of NEDD8 with hepatocyte marker Alb, BEC/progenitor marker Pan-CK, hepatic stellate cell (HSC) marker desmin, and liver macrophage (Kupffer cell) marker F4/80, respectively, in P14 WT and LKO livers. We found a specific reduction of Alb and its colocalized neddylation staining in hepatocytes, confirming hepatocytespecific deletion of neddylation. However, we identified more Pan-CK and NEDD8 double-positive cells as well as more activated hepatic HSCs expressing desmin and NEDD8 in P14 LKO livers, whereas no significant differences in the staining of F4/80, a macrophage marker, and its co-localized NEDD8 were observed between two genotypes. These data confirmed the specific deletion of neddylation in hepatocytes and identified more NEDD8-positive BEC/progenitors and HSCs, which contribute to the recovery of total neddylation levels in P14 LKO livers. The increased number of Pan-CK and NEDD8 double-positive cells also further emphasizes the presence of fetal reprogramming in P14 LKO livers. We have now presented these data in new Supplemental Fig. S4 and described them in the Results section on Page 8.
2. Along these lines of compensatory mechanisms, it would be useful to evaluate the effects of changes in neddylation on ubiquitylation in some more detail. Though the authors allude to it in passing in the discussion, this is especially relevant since the pathways are inter-related for regulation of protein degradation and it is possible that compensatory mechanisms could be at play, especially since it is known that ubiquitin pathways regulate liver disease as well (PMID: 34529948) and interaction between the pathways has been recognized (PMID: 22608973).
Response: Neddylation is well known to target E3 ubiquitin ligases such as CRLs to mediate protein ubiquitination and degradation. Indeed, we have shown dysregulated NRF2 and IκBα expression, two well-known CRL-targeted proteins, in our neddylation-deficient cells or tissues ( Fig. S5e and Fig. S8).
Interestingly, the total ubiquitination levels were upregulated in AAV-Cre livers. Whether such changes are due to the inactivation of deubiquitinating enzymes or alterations of cellular populations remains unknown. Nevertheless, we further assessed the expression of two additional CRL-targeted proteins including p53 and β-Catenin in our ALF model, which further supports our findings that neddylation deficiency dysregulates ubiquitin-mediated proteolysis, contributing to liver pathology. Data are now included as New Supplemental Fig. S10i and described in Results on Pages 16-17.
Meanwhile, PMID: 22608973 reports NEDD8 overexpression results in the neddylation of ubiquitin substrates by the ubiquitin pathway. This phenomenon could happen in many disease states when the NEDD8 level is elevated. Whether NAE1 deficiency causes elevated free NEDD8 leading to atypical neddylation of ubiquitination substrates to alter protein degradation is not known. A comprehensive study of proteome changes in neddylation-deficient livers would require quantitative proteomics, which is out of the scope of this study.
3. The authors suggest that deficiency in neddylation compromises glutathione stores to induce oxidative stress and cell death, since supplementation with NAC protects against these changes to a certain extent.
However, no mechanistic data is provided on why compromised neddylation should cause a decrease in cellular GSH. Some data to confirm mechanisms of this aspect would be useful.

Response:
We apologize for the confusion. In NAE1-deleted livers, the glutathione biosynthetic genes were not downregulated but elevated ( Fig. 4b and Supplemental Fig. S5). This suggests that the reduced GSH level in NAE1-deleted livers is largely attributed to the increased oxidative stress through redox and conjugation reactions. Indeed, we found mitochondrial dysfunction and inflammation are among the mechanisms that cause oxidative stress in NAE1-deleted hepatocytes ( Fig. 4 and   Supplemental Fig. S5). To avoid confusion, we have now reported GSH depletion after the increased oxidative stress in Fig. 4 and Supplemental Fig. S5. We have also modified our discussion accordingly (Discussion, Paragraph 2).
4) The authors show higher steatosis in adult NAE1 deleted livers. However, it was recently demonstrated that inhibition of neddylation ameliorated steatosis in NAFLD (PMID: 34153521), which is opposite of what the authors indicate. This needs to be discussed and possible explanations provided.

Response:
We have now added discussion in Discussion, Paragraph 3, and Page 20. "Conversely, MLN has been shown to stimulate hepatocyte mitochondrial function and fatty acid oxidation to ameliorate steatosis in mouse models of dietary non-alcoholic fatty liver disease (NAFLD). The protective role of MLN was only observed under disease states with higher neddylation levels. Our current evidence suggests that neddylation, although relatively low in healthy adult hepatocytes, is essential for the maintenance of liver homeostasis under physiological conditions. Therefore, its level needs to be carefully fine-tuned when applying neddylation inhibition-based therapies to avoid potential adverse effects on liver function." 5. The authors need to clarify what they interpret from the RIP3K data. Active caspase 8 would cleave RIP3K and induce apoptosis, while RIP3K activation in the absence of caspase 8 functions as a molecular switch for necrosis (PMID: 23913919). However, the authors show increase in both caspase 8 and RIP3K phosphorylation by D24 in Cre mice. However, there seems to be some individual variation since the animal with lower caspase 8 has higher RIP3K and vice versa. These issues need to be clarified.

Response:
We thank the reviewer for pointing that out. Our RIPK3 staining identified necroptotic cells mainly concentrated in zone 3 (Fig. S3a), whereas apoptotic cells were scattered and azonal (Fig. 2e).
Neddylation deficiency also did not trigger necroptosis cell-autonomously (Please see Discussion, paragraph 4). Therefore, caspase 8-mediated apoptosis and RIPK3-mediated necroptosis are occurring simultaneously in D24 Cre livers but not in the same cells. Since D24 Cre livers are very heterogeneous due to massive changes in liver cytostructures, individual variations are expected. 6. What is the basis for the authors statement in the discussion that antioxidants prevent acute liver failure?
While NAC is an antidote to drug-induced liver toxicity such as due to acetaminophen, it functions by replenishing glutathione in that context, rather than scavenging free radicals. Also, NAC prevents liver injury if given early and is unlikely to prevent liver failure if injury is ongoing. The distinction between liver injury and liver failure needs to be made clear throughout the manuscript.

Response:
We appreciate the reviewer's comments. We understand the major role of NAC, as an antidote to drug-induced liver toxicity, is to replenish glutathione. However, the action of NAC results from its antioxidative or free radical scavenging property through increasing intracellular GSH levels. It can also act as a direct scavenger of free radicals. NAC is more effective in treating disease-associated oxidative stress and inflammation 7,8,9 . Especially, NAC treatment did replenish GSH, and reduce ROS and inflammation in AAV-Cre livers. Nevertheless, we have changed antioxidant therapy to NAC therapy.
We highly agree with the reviewer that NAC prevents liver injury if given early and is unlikely to prevent liver failure if injury is ongoing. We found exactly what the reviewer mentioned. NAC therapy is effective only if we provide NAC water at the early onset of NAE deletion (i.e. 10 days after AAV-Cre injection).
Even if we start therapy 2 days later, NAC does not rescue mortality significantly. We have now included this information in the Discussion, Paragraph 2. We have also made distinctions between liver injury and liver failure throughout the manuscript. 7. Page 3, Line 48-The authors state "The mechanisms underlying acute liver injury are not fully understood, especially at the posttranslational level." It is assumed the authors mean acute liver "failure" since mechanisms of injury are well characterized in several systems.
Response: Thank you very much for pointing that out. We have changed it accordingly.
8. Page 5, Line 92: The authors state "This suggests the neddylation pathway may be involved in the pathophysiology of HBV-associated ALF." This sentence is not accurate, since the mere alteration of gene expression in microarray data sets is only that-an alteration. It does not confer a cause-effect confirmation and hence it the authors should modify the sentence.

Response:
We have changed this sentence to "This suggests that the neddylation pathway is perturbed in HBV-associated ALF". Thank you. 9. Were the Nae1fl/fl controls littermates? This needs to be clarified, and if so, specifically stated.
Response: This was originally included in the Results section, Paragraph 2 on Page 5. "Nae1 f/f mice were used as wild-type (WT) controls (Fig. 1a)".